| Clubroot disease cause by obligate plant pathogen (Plasmodiophora brassicae Wor.), is a worldwide disease, which reduces the crop yield and quality, even destruction. The CRb gene was located in 23.67-23.75Mb region of Chinese cabbage A3 linkage group. In this study, map-based cloned method was applied to isolate the CRb gene and identified function. The results are concluded as following:1. Fine mapping of CRb gene and screening the candidate genes:RCA (recessive-class analysis) method was applied to fine mapping the CRb gene. The CRb-linked flanking markers TCR74 and TCR79 were used to screen with 1142 susceptible F2 individuals, thus 44 recombinants were identified between these two markers. Six polymorphism markers including three newly developed markers, two coseparation markers with TCR74 (TCR30 and TCR37) and 1 dominance marker TCR108, were applied to determine the genotype of 44 recombinants. Combined with the resistance test result, the high-density genetic map was constructed, and the CRb gene was located in 0.07cM region, three markers(TCR68、BrG2 and BrG3) coseparation with it. Two genes encode TIR-NBS-LRR protein were assumed to be the candidate genes of CRb, named as CRbl and CRb2.2. CRb candidate gene clone and structure analysis:CRb1 CDS sequence (3630bp) and CRb2 CDS sequence (3669bp) were isolated from resistance material’CR BJN3-2’. CRbl encoded 1209 amino acid and CRb2 encoded 1222 amino acid, both of them were predicted to have the feature domain of TIR-NBS-LRR protein. The CDS and amino acid sequence between resistance and susceptible material showed differences in these two genes. In conclusion, these two genes can be determined the candidate gene further more.3. Expression analysis of CRb candidate genes:Relative expression level of CRb1 and CRb2 were analyzed with leaf, petiole, hypocotyl and root samples with the resistant and susceptible plants, by inoculating the Plasmodiophore brassicae after Oh,12h,24h,36h,72h,4d,6d and 9d. The results indicated that two candidate genes were expressed in all tissues; and relative expression level of them was consistent relatively in the root, as well as there have no law in other three tissues.4. Construction of CRb candidate genes plant expression vectors in Arabidopsis, and functional studies:Construct the CRbl and CRb2 plant express vectors by using the restriction enzyme digestion method, Agrobacterium tumefaciens mediated thansformation obtained 6 CRbl and 12 CRb2 transgenic Arabidopsis lines. Clubroot resistance test were implied with 3 Ti lines for CRbl and 6 Ti lines for CRb2, the result indicated that disease index were both lower in the CRb1 and CRb2 transgenic lines than those of the wild type Arabidopsis.5. Phylogenetic analysis of CRbl and CRb2CRbl and CRb2 amplificated results in 19 clubroot resistant materials and 10 susceptible materials indicated these two genes harbored in A genome of Brassica. Phylogenetic analysis showed that both CRbl and CRb2 have homologs in 19 resistance materials. The homology of CRbl among 19 resistance materials was higher than CRb2, but the homology between resistant and susceptible materials was lower than CRb2.Combined all our data shown above, we successfully identified and characterized 2 functional TIR-NBS-LRR genes (CRbl and CRb2) from CRb locus in Brassica rapa. The relative expression level of them was difference between the resistant and susceptible plants, but has consistent regulatory level in root.Clubroot resistant test showed that, CRbl and CRb2 could play an essential role in the resistance to clubroot disease. Phylogenetic analysis indicated that CRb1 and CRb2 were only existing in the A genome of Brassica, and both CRbl and CRb2 have significant homology among all 19 clubroot resistant materials we collected, these two genes relative conservation in 19 clubroot resistant materials, therefore, they could play an important role in resistant process of clubroot. |
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